β-Lactam antibiotics such as Penicillins and Cephalosporins are widely used for the treatment of a variety of infectious diseases. As Cephalosporins offer better protection than Penicillins particularly against resistant organisms, significant thrust has been given to derivatizations of Cephalosporins to broaden their spectrum and enhance their efficacy. 7-ADCA (7-Amino deacetoxy cephalosporanic acid), 7-HACA and 7-ACA serve as core intermediates for the synthesis of numerous semisynthetic cephalosporins such as Cephradine, Cephalexin, Cephadroxyl, Cefazolin, Cefotaxime, Ceftriaxone, Cefepime, Cefoperazone, Cefuroxime, Cefoxitin and the like. Currently, 7-ACA is derived from Cephalosporin C by the cleavage of the 7-aminoadipoyl side chain by a two stage enzymatic process (U.S. Pat. No. 5,424,196) and 7-ADCA is derived from the hydrolysis of phenylacetyl-7-ADCA by Penicillin G amidase, wherein the phenylacetyl-7-ADCA is manufactured by conventional chemistry of ring expansion of Penicillin G. 7-ADCA or ceph G being cheaper than 7-ACA or Ceph C respectively, a process to viably convert 7-ADCA with or without a substituent at 7-amino position, like phenyl acetyl group, to the corresponding deacetyl cephalosporanic acid, would provide a cheaper alternate to HACA.
Native Penicillins and Cephalosporins are produced by a variety of bacteria and fungal organisms and significant progress has been made in understanding their regulatory architecture (Aharonowitz, Y., et al., Annu. Rev. Microbiol. 46: 461-495, 1992; Axel A. Brakhage, Microbiol. Mol. Biol. Rev. 62: 547-585, 1998). Three amino acids, L-valine, L-α-amino adipate and L-cysteine condense to form the tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-Valine which then, gets cyclized to form isopenicillin N. In the case of penicillin producers, isopenicillin N gets converted to penicillin G, penicillin V, etc. The biosynthesis of cephalosporin by epimerization of isopenicillin N to penicillin N in organisms such as C. acremonium, S. clavuligerus, etc leads to cephem compounds such as cephalosporin C and Cephamycin C. The rate limiting or rather, the committed step in the biosynthesis of Cephalosporins is the expansion of the five-membered thiazolidine ring of Penicillin N to a six-membered dihydrothiazine ring of deacetoxy Cephalosporin C (DAOC). In prokaryotes such as Streptomyces clavuligerus, the ring-expansion step of Penicillin N is catalyzed by an enzyme called deacetoxy cephalosporin C synthase (DAOCS or cefE or expandase) and hydroxylation of DAOC is carried out by cephalosporin hydroxylase (deacetyl cephalosporin C synthase or DACS or cefF or hydroxylase) (Jensen S. E. et al., Journal of Antibiotics, 38, 263-265, 1985). In eukaryotic organisms such as Cephalosporium acremonium, both of these reactions are catalysed by a single bifunctional enzyme DAOCS-DACS (expandase-hydroxylase or cefEF) (Dotzlaf, J. E. and Yeh, W. K., Journal of Bacteriology, 169, 1611-1618, 1987). Subsequently, deacetyl cephalosporin C (DAC) gets acetylated to Cephalosporin C in Cephalosporium acremonium, while in bacteria such as Streptomyces clavuligerus, further modifications occur resulting in Cephamycin C.
The cefEF gene that codes for 332 amino acids encodes the expandase-hydroxylase in C. acremonium (Samson, S. M., et al., Nature Biotechnology, 5, 1207-1215, 1987).
The deacetyl cephalosporin C synthase gene (cefF) has been cloned and sequenced from S. clavuligerus and it encodes 318 amino acids (Kovacevic, S. and Miller, J. R., Journal of Bacteriology, 173, 398-400, 1991). The hydroxylase has 54% amino acid sequence identity with that of expandase-hydroxylase of C. acremonium. The expandase, expandase-hydroxylase and hydroxylase enzymes are iron (II) and α-ketoglutarate dependent oxygenases and they are part of a subfamily of the mononuclear ferrous enzymes.
Penicillin N is the natural substrate for expandase and expandase-hydroxylase and DAOC is the substrate for hydroxylase. However, altered substrate specificity has been detected for expandase-hydroxylase for different substrates such as Penicillin G, Penicillin V, 6-α-Methylpenicillin N and adipoyl-6-APA (Lloyd et. al. Journal of Biological Chemistry, 279, 15420-15426, 2004). As a result, development of green technologies for the manufacture of Cephalosporin intermediates, thus, narrowed down to these enzymes. However, these enzymes show poor capability to convert readily available substrates such as phenylacetyl-7-ADCA and hence, engineering them for commercial applications is required. WO 2008/107782 (445/CHE/2007) describes such manipulations for hydroxylase and its use in the bioprocess for the preparation of compound of formula (I), some of the strains obtained according to this patent like MTCC 5739, MTCC 5741, MTCC 5746 to MTCC 5749 are not industrially scalable due to the poor hydroxylase activity and hence still it is required to identify better hydroxylase mutant in view of industrial production.
Also in publications like Baker, B. J., et al., Journal of Biological Chemistry 266, 5087-5093, 1991; Coque, J. J. R., et al., Applied Microbiology and Biotechnology 44, 605-609, 1996, conversion of 7-aminoadipoyl deacetoxy cephalosporanic acid to the corresponding deacetyl cephalosporanic acid has been described. When tested these methods did not convert more than 5% of the substrate at very low concentrations. These methods were thus not practical and scalable. No prior art discloses a scalable method for hydroxylation of deacetoxy cephalosporins.
EP 465189 provides the DNA compounds that encode hydroxylase activity of S. clavuligerus. This patent discloses the method for expressing hydroxylase activity of S. clavuligerus in a recombinant host cell. The DNA compound that encodes hydroxylase activity was isolated form S. clavuligerus genomic DNA and it is used to construct recombinant DNA expression vector called pOW399. The cloned hydroxylase gene is used in the hydroxylation of cephalosporin compounds; however the activity of enzyme is unsatisfactory for industrial manufacturing.
U.S. Pat. No. 6,180,361 deals about DNA compounds, recombinant DNA cloning and expression vectors that encode DAOCS and DACS activity. The DNA compound that encodes the DACS/DAOCS activities was isolated from Cephalosporium acremonium genomic DNA and used to construct recombinant DNA expression vectors. This invention also discloses a method for expressing a Cephalosporium deacetoxycephalosporin C synthetase/hydroxylase polypeptide in a recombinant host cell.
Hence there is a need to identify a cephalosporin hydroxylase with enhanced activity for hydroxylation of compound of formula (II), which is cost effective and industrially scalable. As native cephalosporin hydroxylase shows poor affinity for compound of Formula (II) and industrial utility demands development of modified hydroxylase to enhance its activity. As a result, current invention is related to the development of modified hydroxylase from S. clavuligerus having increased substrate specificity for substrates such as compound of Formula (II), when compared with the wild-type hydroxylase. Current invention also provides a scalable process for the hydroxylation of deacetoxy cephalosporanic acid catalyzed by the modified cephalosporin hydroxylase or any α-ketoglutaric acid dependant hydroxylase.